豆类丝核菌中生物碱的提取及苦马豆素含量测定

Extraction of Alkaloid from Rhizoctonia leguminicola and Determination of Swainsonine Concentration

将豆类丝核菌菌株 7-1、7-3接种于改良 Czapek′s培养基 ,置 2 5～ 2 7℃培养箱培养 1 4d。收集菌丝体 ,自然干燥后乙醇索氏提取 ,回收乙醇至糖浆状 ,H2 O∶ CH2 Cl2 (3∶ 1 ,v/v)萃取 ,水层调 p H1 0后 ,再用 H2 O∶CH2 Cl2 (3∶ 1 ,v/v)萃取 ,水层调 p H7,浓缩后上 73 2型强酸性阳离子交换树脂 ,用 1 mol/LNH4 OH洗脱 ,收集氨洗液 ,调 p H7,浓缩后冻干 ,得到一种浅黄色粉末。取苦马豆素标准品 1 0 0 μg及浅黄色粉末提取物 1 0mg,分别用 1 ml吡啶溶解 ,取 0 .1 ml加 BSTFA0 .1 ml,5 0℃水浴 3 0 min。取反应液 1 μl,直接进样作 GC。结果表明 ,在相同的气相色谱条件下 ,在相同出峰时间 (1 .2～ 8min)内 ,标准品的峰形与总生物碱的峰形完全一致 ,可以判定生物碱中含有苦马豆素。根据计算得出菌株 7-1、7-3生物碱中苦马豆素含量为 5 .90 3 mg/g和 7.0 0 6mg/g。

Strains 7-1 and 7-3 were cultured on Czapek′s medium at 25～27℃ for 14 d.Collected mycelium,air dried, and extracted with ethanol(Soxhlet,24hr).The extract was concentrated to a syrup,shaken(3×) with H 2O∶CH 2Cl 2( v/v ),the water extract was brought to pH 10 and again extracted(3×) with H 2O∶CH 2Cl 2( v/v ).The water layer was brought to pH 7 and absorbed(30 min) on a strong cation exchange resin(H +),eluted with 1 mol/L NH 4OH, and the filtrate was dried.The residue was brought pH 7 and lyohilized to obtain a light brown powder.After dissolved 100 μg standard sample and 10 mg power in 1 ml pyridine,0.1 ml of the pyridine was applied to GC.The powder contains swainsonine,for the peak of the powder was consistent with the standard sample at the same GC condition.The peak of strain 7-1 was 116.75 mm(n=10),strain 7-3 was 138.56 mm(n=10),the standard sample was 197.78 mm (n=10)?The concentration of swainsonine in powder of 7-1 and 7-3 were 5.903 mg/g and 7.006 mg/g respectively.